BiotechnologyBreeding interface for Enhancement of Nutritional Quality in Cereals

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Biotechnology-Breeding interface for
Enhancement of Nutritional Quality in Cereals
P.K.Agrawal H. S. Gupta VPKAS, Almora
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• Why micronutrients are important?
• Genetic enhancement feasible?
• Methods available?
• VPKAS for Hill Agriculture beyond?

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Why Need?

• Cereals like rice, wheat and maize are the staple
  food
• In Developing countries, cereals make the major
  part of the diets
• Developing countries suffer from under-nutrition
  and mal-nutrition
• Breeding could help to reduce the problem

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In Developing countries,

• Mineral Vitamin deficiencies affect more people
  than protein-energy malnutrition
• Hidden hunger affect more than hunger per se
• Indonesia mortality rate was observed to be
  reduced by 34 when Vit A was administered

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WHO report

• ½ million child go blind because of Vit A
  deficiency
• gt 3 billion people are iron deficient
• In India alone, Fe fortification to ½ of pregnant
  woman will cost US 37 million
• Breeding for high Fe, Vit A, P, and other
  micronutrients is an novel option

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Is Breeding viable?

• Zn dense varieties in Australia
• Iron efficient Soybean
• Rice accessions with high Fe
• Microefficient plants in Micronutrient
  defficient soils
• Bioavailability of that extra micronutrients

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Successful Examples

• Opaque 2 locus in maize
• lpa mutants in maize, rice and barley
• Golden rice
• Rice Germplasm

Basmati rices are rich in Fe, Zn, and many
minerals
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Methods

• Marker based approach
• Trait available within the pri-/ sec gene pool
• Crossable and transferable
• Transgenic approach
• Trait comes from a distant genome
• Cloned gene is available
• Method of transformation

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Marker based approach at VPKAS

• QPM in Maize
• Low Phytate in maize
• MAS for blast resistance in rice

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Marker Assisted Selection Fast Track Breeding

• phi057, umc1066 and phi112 three SSRs located
  within opaque2 An ideal marker system
• opaque2 a recessive gene and phenotype
  determination requires biochemical assay
• MAS shortens the breeding cycle and accelerates
  the pace of development of an high yielding
  variety/hybrid
• No need for extensive biochemical analysis
• Marker aided background selection reduces linkage
  drag significantly

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Steps in MAS for QPM
Parental Polymorphism Analysis between Normal and
QPM lines Foreground selection using opaque2
specific SSRs in BC1 and BC2 Background
Selection using genome-wide SSRs in
BC2 Phenotypic selection for hard endosperm
trait in marker selected BC2F2 plants
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Polymorphism Analysis for opaque2 b/w QPM and
Normal Inbreds
umc1066
phi057
phi112
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Foreground Analysis for opaque2 in BC1 using
umc1066
Genotyping backcross (BC1) progenies of QPM X non
QPM crosses using umc 1066. Double bands indicate
desirable heterozygotes and the single bands
indicate discarded homozygotes
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Foreground Selection in BC2F2 using umc1066
Identification of homozygous recessive
individuals in BC2S2 generation employing umc
1066. The first two lanes correspond to non QPM
(P1) and QPM (P2) parents while rest of them are
individuals in BC2S2 population. The individual
plants indicated by are homozygous for
recessive opaque 2 mutant allele
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Background Selection Rapid Recovery of RPG
Generation BC2 A genome 95.9 B genome 4.1
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Phenotypic Selection for Endosperm Modification
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FQH 4567
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Strategy for FQH 4567
CM 212
CM 145
X
VQL 1
VQL 2
FQH 4567
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Varieties under consideration for conversion into
QPM version and their present status

• QPM donor parents CML189, CML180 CML 173
• Recipient parents CM141, V351, V353, V354, V359
  V360 in BC2 stage.
• Background selection is in
  progress
• Recipient parents V364,V366, V368, V370, V371,
  V372, V373 V374 in BC1 stage.

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Transgenic approach

• Maize Transformation
• Rice transformation

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Maize transformation
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Golden Rice increased Vitamin A content (but
not without controversy) transgene three
pathway enzymes
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?-Carotene Pathway Problem in Plants
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The Golden Rice Solution
?-Carotene Pathway Genes Added
Daffodil gene
Single bacterial gene performs both functions
Daffodil gene
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Novel transformation methods
should have

• Co-transformation (multiple genes)
• Simple integration pattern
• Higher co-expression
• Antibiotic-marker free
• Independent segregation
• Stable over generations

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Strategy for generating transgenic plants
Digestion with restriction enzymes
Minimal linear cassettes
Whole plasmids
Bombardment (cotransformation)
Selection and Regeneration
Growth
Transformed callus
Regenerating callus
Transgenic plants (with multiple cassettes)
Agrawal et al., 2000, 2005 Fu et al, 2000 Loc et
al, 2003
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Minimal cassettes used
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Integration pattern
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Advantages of Clean DNA Transformation

• Co-transformation
• Higher co-expression
• Simple integration pattern
• Stable over generations
• Independent segregation

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Anthranilate synthase in monocots
as- from Ruta species

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Anthranilate synthase in monocots
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Study life better it in a holistic approach
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Thanks